PH-Responsive Polyethylene Glycol Monomethyl Ether-ε-polylysine-g-poly(Lactic Acid)-based Nanoparticles As Protein Delivery Systems

Objective:With good biocompatibility and easy degradation,poly（lactic acid）（PLA）is widely used in development of protein drug delivery system.In recent years,more attention was paid to PLA nanoparticles for drug delivery.The application of PLA for sustained protein delivery is restricted by the harsh pH inside carriers,which leads to inactivation of the loaded proteins.This study aims to synthesize a pH-responsive comb-shaped block copolymer,polyethylene glycol monomethyl ether-ε-polylysine-g-poly（lactic acid）（PEP）to deliver protein[bovine serum albumin（BSA）].Methods:We synthesized the functional copolymers（PEP）through a typical amide reaction and detected the structure of the copolymers with ¹H NMR.The polydispersity and molecular weight of PEP were detected with gel permeation chromatography（GPC）.We employed nanoprecipitation to prepare nanoparticles（NPs）loading BSA and blank nanoparticles without BSA.Transmission electron microscopy（TEM）was used to examine the shape of NPs.Zetasizer 3000HS was employed to measure the zeta potential of the NPs.X-ray photoelectron spectroscopy（XPS）was used to examine the superficial chemical components of NPs with PEG of different molecular weight.Water absorption and degradation of the NPs and in vitro BSA release and kinetics were investigated.We studied the cell viability of HL-7702cells which were treated with PEP52 NPs of different concentrations.Acute toxicities of PEP NPs and EPL were assessed in Kunming mice through tail vein administration.The microclimate pH inside PEP NPs was mapped with ratiometric method.The stability of BSA released from PEP NPs was investigated with circular dichroism（CD）spectroscopy.In vivo distribution and pharmacokinetics of NPs loading BSA were studied in Kunming mice after tail vein administration.Results:¹H NMR spectrum of PEP confirmed the content of PLA and EPL in the copolymer.The transmission electron micrographs showed that PEP NPs present with fine spherical shape and no aggregation was observed.XPS examination showed that the azote content was decreased with the increase of PEG molecular weight.No azote content was detected when PEG molecular weight was higher than 5 kDa.These findings suggested that PEG segments locate on the surface of NPs,and PLA intertwining with EPL formed the NP core.PEP NP zeta potential was reduced from48.62mV to 21.17mV with increased PEG molecular weight（0–10 kDa）,and size was enlarged from 84.55 nm to 130.57 nm.The above findings confirmed that PEG segments overlaid the core.The water uptake of the PEP52 NPs was 112.7%,which was higher than that of the PLA NPs（101.8%）.The water absorption of the NPs increased from 110.8%to 135.1%with increased PEG molecular weigtht[2 kDa（PEP22）to10 kDa（PEP102）]in PEP.PLA NPs had an obvious initial burst release during the first 48 h.However,after 48 h,PLA NPs released less amount of BSA during the 384 h period than any PEP NPs.MTT assay showed that on the 4^thh day,the cell viability was higher than 80%in the group treated with PEP NPs of different concentrations.No obvious toxic effect was observed with the increase of PEP NPs concentration.No abnormality was found in the livers,spleens,and kidneys of mice treated with PEP NPs after 14 days.The microclimate pH levels of the PLA NPs decreased gradually with time because of the products of acidic degradation.Nevertheless,the pH inside PEP52 NPs was relatively constant（pH range:7.10–7.37）at 32 days.The release curve of BSA from PEP52 NPs on days 2,4,8,16,and 32showed obvious wave hollow at 208 nm and 220 nm,with estimated helix contents of58.3%,57.9%,56.7%,52.4%,and 50.8%.In contrast,PLA NPs released protein after32 days presented lowerα-helix contents compared with that released from PEP52NPs.The estimatedα-helix contents on days 2,4,8,16,and 32 were 54.1%,51.6%,42.5%,36.7%,and 30.8%,respectively,with significant differences compared with PLA NPs（P<0.05）.These findings suggested that PEP52 NPs could effectively protect BSA and prevent it from inactivation.The fluorescence levels in the blood and organs of the mice treated with PEP52 NPs were significantly higher than those obtained after the administration of PLA NPs,EP02（ε-polylysine-g-PLA）NPs,and BSA-FITC（P<0.05）.In vivo observation of BSA loaded NPs in Kunming mice showed that PEP52 NPs exhibited longer circulation time（P<0.05）.The AUC_{0–384 h}–384 h values of PEP52 NPs were 1136.978,986.809,and 603.238μg/g in the liver,spleen,and kidney,respectively,indicating its clearance in the order of liver,spleen,and kidney.Conclusions:As a protein delivery system,the PEP NPs synthesized in this study can provide an appropriate pH microclimate for the sustained release of BSA.The PEP NPs showed good biocompatibility and prolonged circulation time,suggesting its application potential for controlled release of bioactive proteins.